Recombinant proteins have proven to be medically useful therapeutic agents for the treatment of a wide variety of diseases ranging from bleeding disorders, to diabetes, cystic fibrosis, cancer, and viral infections. The ability to produce biologically active recombinant protein therapeutics in large amounts and at lower cost is very often confounded by improper folding and extensive aggregation of many potentially useful proteins, particularly those that contain disulfide bonds. The long-range goal of this research is to provide more effective strategies that mimic the molecular mechanisms used by cellular folding catalysts and chaperones to promote efficient protein folding. The immediate experimental goals are to investigate the multiple mechanisms for assisting protein folding that are used by protein disulfide isomerase (PDI), a major protein folding catalyst of the eukaryotic endoplasmic reticulum. Functionally, PDI catalyzes slow disulfide bond formation and rearrangement during protein folding, inhibits the aggregation of unfolded proteins at high concentration (chaperone activity), and has the unusual ability to actively increase the aggregation of some unfolded proteins at low concentration (anti- chaperone activity). In addition, PDI is known to bind hydrophobic molecules such as estrogen and thyroid hormone (T3). Experiments using site-directed mutagenesis coupled with the tools of biochemistry are proposed to examine the spatial, structural and functional relationships among the various active sites involved in PDI's interaction with unfolded proteins, thee mechanisms PDI uses to gain access to buried thiols in stabilized folding intermediates, and the contribution of intermolecular reactions to PDI catalysis. The mechanisms of PDI's unusual chaperone/anti-chaperone activities and the mechanisms of protein aggregation in general will be investigated by kinetic and physical studies of aggregation including dynamic light scattering and turbidity measurements, examination of aggregate composition and stability, and an investigation of the specificity involved in the formation of heterogeneous aggregate involving multiple proteins. Catalyzed protein folding and protein aggregation are not well-understood at the molecular level. The proposed studies will provide an important step toward realizing the long-range goals of improved folding and production methods for recombinants proteins of medical importance.